Squamous cell carcinoma of the head and neck (HNSCC) is the 6th most common cancer in the developed world, affecting nearly 44,000 patients each year in the US, which results in ~11,000 deaths. The vast majority of these malignancies involve neoplastic lesions in the oral cavity, lip, and pharynx. Like most cancers, HNSCC results from a series of discrete, irreversible and sequential alterations in genes that control cell growth and differentiation, together with genetic aberrations promoting invasion and metastasis. The goal of our program is scientific excellence in addressing the devastating problem of oral cancer. In particular, we aim to elucidate the molecular changes that contribute to the evolution of oral neoplasia, and to use this knowledge to develop markers of disease progression and novel therapeutic approaches for oral malignancies. Genomic and proteomic approaches to understand oral cancer. Our laboratory has used a number of novel approaches to investigate gene and protein expression profiles in HNSCC. We began by demonstrating the successful use of laser capture microdissection (LCM) to procure specific cell populations from heterogenous clinical samples of HNSCC, and showed that LCM-procured material can be used effectively to extract RNA, DNA, and proteins. In collaboration with Myung Hee Park?s group, we have studied gene expression profiles in normal and immortalized human oral keratinocytes, and in a large collection of distinct HNSCC cell lines. Genes involved in cell cycle regulation, cell proliferation, apoptosis, differentiation, and cell adhesion were widely altered in cancer cells, thus providing substantial background information on genes that are differentially expressed in normal and malignant squamous cells. Hierarchical clustering analysis and principal component analysis revealed two distinctive subtypes of gene expression patterns, which reflect a degree of heterogeneity in HNSCC. As part of a long term collaboration with Dr. A. Yeudall (VCU, VA), we have used a panel of cell lines exhibiting normal and constitutive activity of the epidermal growth factor receptor (EGFR), to investigate which of the genes activated by EGF stimulation in normal keratinocytes are aberrantly expressed in HNSCC cells. We extended this analysis with the use of cell lines derived from primary and metastatic HNSCC tissues. This approach enabled the identification of an EGFR-trancriptional signature, which we are now investigating further. Ultimately, the aberrant expression and activity of molecules present in HNSCC cells are responsible for their malignant behavior. Thus, we have also made a concerted effort to investigate protein expression profiles during HNSCC tumor development. For example, we have examined the activity of tissue remodeling proteases in HNSCC in collaboration with PTRU, and conducted a direct proteome-wide analysis of LCM-procured normal and tumor tissues using a combination of liquid chromatography (LC) and tandem mass spectrometry (MS). In collaboration with William Hancock (Barnett Institute, MA), techniques for protein extraction and global proteolysis of whole cell lysates, followed by the separation of complex peptide mixtures by reverse phase LC and analysis by MS followed by tandem MS sequencing of selected peptides were optimized. In an initial analysis of clinical samples from HNSCC patients, amininoacid sequence information was obtained from 94-105 proteins per tissue set. Further development of these techniques may allow proteome-wide profiling of HNSCC tissues. Dysregulated signaling networks in HNSCC: novel mechanism-based approaches for HSNCC treatment. Recent findings suggest that the ability of Akt to coordinate mitogenic signaling with nutrient-sensing pathways controlling protein synthesis may represent an essential mechanism whereby Akt ultimately regulates cell growth. This pathway is initiated by Akt phosphorylation and inactivation of a tumor-suppressor protein, tuberous sclerosis complex protein 2 (TSC2), which is also known as tuberin. TSC2 associates with a second tumor-suppressor protein, tuberous sclerosis complex protein 1 (TSC1), and act together as a GTPase activating protein (GAP) for the small GTPase Rheb1. Thus, inactivation of TSC2 by Akt leads to the accumulation of the GTP-bound (active) form of Rheb1, which in turn promotes the phosphorylation and activation of an atypical serine/threonine kinase known as the mammalian target of rapamycin (mTOR). mTOR then phosphorylates key eukaryotic translation regulators, including p70-S6 kinase (p70S6K) and the eukaryotic translation initiation factor 4 E binding protein 1 (4E-BP1). The latter prevents the repressing activity of 4E-BP1 on the eukaryotic initiation factor 4E (eIF4E), ultimately resulting in enhanced translation from a subset of genes that are required for cell growth. Of direct relevance to HNSCC, eIF4E gene amplification and protein overexpression is often associated with malignant progression of this cancer type, and its expression levels in surgical margins can predict tumor recurrence. In a recent study, we showed that the Akt-mTOR pathway plays a central role in HNSCC. Initially, we observed that the aberrant accumulation of the phosphorylated active form of S6 (p-S6), the most downstream target of the Akt-mTOR-p70S6K pathway, is a frequent event in clinical specimens from HNSCC patients and in a large panel of HNSCC-derived cell lines. We also found that the level of activated S6 was rapidly reduced when HNSCC cell lines were treated with rapamycin, which specifically inhibits mTOR. Furthermore, p-S6 was dramatically reduced in HNSCC xenograft models at clinically relevant doses of rapamycin. Concomitantly, we observed that rapamycin exerts a potent anti-tumor effect in vivo, as it inhibits cell proliferation and induces apoptotic cell death of HNSCC cells ultimately promoting tumor regression. These findings identified the Akt-mTOR pathway as a potential therapeutic target for HNSCC, thus raising the possibility of exploring the clinical activity of rapamycin and its analogs in HNSCC patients. Development of conditional animal models for squamous carcinogenesis. With the assistance of GTF, NIDCR, we have generated transgenic mice carrying the tetracycline-inducible system (tet-on receptor) targeted to the basal layer of stratified epithelium using the cytokeratin 5 promoter. In this system, transgenes of interest, including candidate oncogenes, can be expressed under the control of a tetracycline-responsive promoter. As a proof of principle, we have examined the ability of active ras genes to promote SCC when conditionally expressed. Although prior animal models suggested that active ras alone promotes benign papilloma formation with a variable rate of delayed carcinoma conversion, the use of this system, which targets a cell compartment that includes the epidermal stem cells, was sufficient to promote the rapid (10-20 days) formation of malignant SCC in the skin and oral tissues of animals treated with doxycycline. Using this model, we are now positioned to test the transforming potential in vivo of activated alleles of newly discovered signaling molecules, alone or in animals engineered to be deficient in relevant tumor suppressor genes. Current and future use of this model system may help to unravel the mechanisms responsible for SCC, and aid in the search for alternative oral cancer treatments.